1. Field of the Invention
The present invention relates to a method and apparatus for distinguishing multiple subpopulations of particles, and more particularly, concerns a method and apparatus for simultaneously distinguishing and enumerating multiple subpopulations of cells which have been labeled with different fluorochromes.
2. Description of the Prior Art
Presently known and available flow-through cytometers and the like particular detecting devices commonly include two channels for the detection of two subpopulations of cells in a mixture. For example, devices are known which include two fluorescence channels which can detect cells specifically labeled with two fluorescent agents associated with the respective fluorescence channels. In these type devices, a complete fluorescence channel including the electrical circuitry and fluorescence detectors has been required for each fluorochrome-treated cell to be detected in the mixture of cells in the sample being analyzed. Therefore, in order to detect multiple subpopulations of cells in a sample using flow-through cytometry, an equivalent number of fluorescence channels is required using the known, conventional devices. A further limitation is that the nature of excitation and emission characteristics of fluorochromes makes it difficult to acquire more than two fluorochromes, attachable to protein, which provide emissions sufficiently separated in wavelength. Some representative devices using conventional flow-through cytometry are described in U.S. Pat. Nos. 4,198,160; 3,738,759; 3,864,571; and in "A Proposal for an Automatic Multi-parameter Analyzer for Cells (AMAC)," by Robert C. Leif, Automated Cell Identification and Cell Sorting, edited by George L. Wied, Academic Press, New York 1970, pages 131-159.
There are many instances when it is desirable to be able to detect multiple subpopulations of cells from a sample mixture. However, as alluded to above, one of the disadvantages found in conventional equipment is that a plurality of fluorochromes would have to be employed for labeling the cells, as well as an equivalent number of fluorescence channels to monitor the specific spectral characteristics associated with the individual fluorochromes. Moreover, a sufficient plurality of fluorochromes is not presently available. Clearly, this has created formidable problems. While it is desirable to be able to detect, and also enumerate, multiple subpopulations of cells from a sample mixture, it is even more desirable to minimize the number of fluorochromes employed as well as the number of fluorescence channels and the associated circuitry. With this in mind, the present invention is directed to solving the aforementioned problem, while satisfying the desired need for the determination of multiple subpopulations of cells from a sample mixture.